REPLI-g WTA Single Cell Kit

For whole transcriptome amplification of total RNA or mRNA from single cells

Complete transcriptome coverage from just single cells

Uniform WTA with negligible sequence bias due to MDA technology

Optimized for use with new technologies, including NGS

Amplification of total RNA or mRNA-enriched (poly A+) RNA

Novel tool for cancer and stem cell research

The REPLI-g WTA Single Cell Kit enables reliable investigation of effects on transcription regulation at the single-cell transcriptome level and allows uniform amplification of all transcripts from just single cells (1–1000 cells). Dedicated buffers and reagents undergo a unique, controlled decontamination procedure to block amplification of contaminating nucleic acids by the REPLI-g method. The innovative lysis buffer effectively stabilizes cellular RNA, ensuring the resulting RNA accurately reflects the in vivo gene expression profile. All enzymatic steps have been developed to enable efficient processing of RNA for accurate amplification of cDNA, which is achieved with negligible sequence bias using innovative Multiple Displacement Amplification (MDA) technology.

The REPLI-g WTA Single Cell Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

High number of mappable reads from just 3 cells.

REPLI-g WTA Single Cell reactions were performed on 3–1000 cells in various replicates, using the mRNA (poly A+) enrichment protocol to reduce rRNA amplification. WTA amplified cDNA was fragmented (Covaris S220) and an NGS sequencing library was prepared using the GeneRead Library Prep I Kit (QIAGEN). Sequencing was done on a MiSeq Instrument (Illumina) and RNA biotypes were mapped using Bowtie2. Results demonstrate that the majority of reads (>80%) are mappable to protein coding RNA and Linc RNAs, and that a negligible number of reads map to other, non-targeted RNA biotypes (data for minor RNA biotypes not shown). Comparable results between values obtained after the sequencing of all WTA samples (Mean value [all cells]) and 3-cell WTA samples (Mean value [3 cells]) were obtained.

High level of experimental reproducibility.

[A] REPLI-g WTA Single Cell reactions were performed using the mRNA (poly A+) enrichment protocol to reduce rRNA amplification. WTA amplified cDNA was prepared as described in "High number of mappable reads from just 3 cells" and sequenced on a MiSeq Instrument (Illumina). RNA biotypes were mapped to single-transcript RNA using Bowtie2 and reads per kilobase and million mapped reads (RPKM) were calculated. Results demonstrate comparable average RPKM values of the 3-cell samples versus transcripts derived from WTA samples (10–50 cells). [B] REPLI-g WTA Single Cell reactions, including rRNA amplification, were performed on individual human cells. Real-time PCR of various transcripts (18S rRNA, 28S rRNA, ddx5, beta-actin, HPRT, GAPDH, PPIA, c-myc, RPS27a, BANF-1, abl-1) was done using QuantiFast SYBR Green PCR reagents and 1 ng of WTA-cDNA. CT values normalized to 18S rRNA from two individual single cell WTA reactions demonstrate a high level of concordance in RNA amplification between experiments, with a high R2 value of >0.97.

Highly sensitive detection of even low-abundance transcripts from single cells.

Whole transcriptome amplification was performed from 15 different single cells or 15 replicates of 10 pg total RNA using the REPLI-g WTA Single Cell Kit. Real-time PCR analysis of 1 ng WTA-amplified cDNA from a variety of different transcripts was done to quantify high-, medium-, and low-copy transcripts. [A] Box plots were calculated from ΔCT (CT [WTA-DNA] – average CT [WTA-DNA]) to differentiate biological differences from the method-derived technical noise. The green box indicates increased gene expression and the red box indicates decreased gene expression compared to the mean. Technical noise, representing the limit of sensitivity, is depicted by the white box, which is clearly less than the biological differences detected. [B] Analysis of a low-copy transcript (abl-1) in 15 individual cells demonstrates the variability of transcript levels between individual cells and that most CT values are outside the low level of technical noise (depicted by black lines). These findings indicate that the REPLI-g WTA Single Cell Kit is highly suited to analyze even low-abundance transcripts from just single cells.

More sensitive detection of even low-abundance transcripts.

Whole transcriptome amplification was performed using 20 pg of total RNA. [A] Unlike kits from other suppliers, which were less successful at amplifying the same amount of the same transcript, 100% of high-, medium-, and low-abundance transcripts were detected following RNA amplification using the REPLI-g WTA Single Cell Kit. [B] Real–time PCR of 22 medium-abundance transcripts, representing the medium-abundance transcript row in [A], demonstrated that only the REPLI-g WTA Single Cell Kit reliably amplified all transcripts. Transcripts that could not be detected gave CT values >40 (red bar).

REPLI-g amplified cDNA performs like gDNA in downstream experiments.

REPLI-g Kits amplify genomic DNA or RNA from a wide variety of sample types, generating amplified DNA and cDNA that performs just like gDNA and is highly suited for numerous downstream experiments.

Multiple Displacement Amplification (MDA) technology.

Primers (arrows) anneal to the template DNA and are extended at 30ºC by REPLI-g SensiPhi DNA Polymerase, which moves along the cDNA template strand, displacing the complementary strand while becoming a template itself for replication. In contrast to PCR-based amplification, MDA does not require different temperatures and ends in very long fragments with low mutation rates.

REPLI-g WTA Single Cell Kit procedure.

A single cell sample (containing 1–1000 cells) is lysed efficiently within 5 minutes, with no effect on RNA integrity. Following cell lysis, gDNA is removed prior to the WTA process. Depending on the primer chosen during the subsequent reverse transcription reaction, all transcripts (if performing total RNA enrichment using random and oligo dT primers) or only poly-adenylated transcripts (if performing poly A+ mRNA enrichment using oligo dT primers) will be amplified. The synthesized cDNA is ligated in a high-efficiency ligation reaction. Ligated cDNA is then amplified utilizing MDA technology with the novel REPLI-g SensiPhi DNA Polymerase in an isothermal reaction lasting 2 hours.

Performance

Complete transcriptome coverage, with low experimental variability

The REPLI-g WTA Single Cell Kit contains novel REPLI-g SensiPhi DNA Polymerase, as well as an optimized set of buffers and reagents for whole transcriptome amplification (WTA) from just single cells, up to 1000 cells, or equivalently small samples. Following efficient cell lysis, complete removal of genomic DNA (gDNA), and sensitive reverse transcription, the kit utilizes Multiple Displacement Amplification (MDA) to uniformly amplify cDNA across the entire transcriptome with negligible sequence bias (see figure Multiple Displacement Amplification (MDA) technology). cDNA amplified using the REPLI-g WTA Single Cell Kit demonstrates a high degree of reproducibility from cell to cell and experiment to experiment, in both next-generation sequencing (NGS) and in real-time PCR analysis of specific transcripts (see figure High level of experimental reproducibility). The amplified cDNA can be easily used in a variety of downstream applications (see figure REPLI-g amplified cDNA performs like gDNA in downstream experiments).

Significant number of reads map to protein-coding RNA

The ability to amplify mRNA-enriched RNA (poly A+) makes the REPLI-g WTA Single Cell Kit particularly suited for use in NGS to investigate effects on transcription regulation at the single-cell transcriptome level. Amplification of ribosomal RNA (rRNA), which generates more than 90% of NGS reads, is virtually eliminated, allowing generation of meaningful mRNA-Seq data. Following WTA using an mRNA-enrichment protocol, more than 80% of reads map to protein coding RNA (see figure High number of mappable reads from just 3 cells).

Reliable detection of low-abundance transcripts

Single cell analysis can be challenging when transcript abundance varies greatly within a cell. For accurate results, it is essential that whole transcriptome amplification reliably amplifies all transcripts, regardless of their levels within the cell. The REPLI-g WTA Single Cell Kit is highly suitable for the analysis of even low-abundance transcripts from just single cells. Real-time PCR analysis of the REPLI-g amplified transcript abl-1 demonstrates that even low-copy transcripts can be reliably detected following amplification using the kit (see figure Highly reliable detection of even low-abundance transcripts from single cells). Unlike kits from other suppliers, the REPLI-g WTA Single Cell WTA Kit allows sensitive detection of all transcripts, from high- to low-abundance (see figure More sensitive detection of even low-abundance transcripts).

For use in a wide variety of applications and research areas

The REPLI-g WTA Single Cell Kit efficiently generates and amplifies cDNA from single cells, such as tumor cells, stem cells, or sorted cells and from purified total or poly A+ RNA (10 pg – 100 ng), making the kit highly suited for a wide variety of research areas (see table).

Range of sample material and research areas

Sample material (cells/total RNA)

Research area

Human/animal

Biomarker research (expression)

Stem cell research

Analysis of circulating fetal cells

Mosaicism studies

Genetic predisposition studies

Typing of transgenic animals

Cancer

Somatic genetic variant analysis

Tumor progression

Tumor stem cells/evoluation

Analysis of circulating tumor cells

Principle

Regulation of transcription is driven by a variety of influences, such as stress, cellular environment, or by disease or somatic genomic variation (e.g., point mutations, copy number variations, or structural variations). Additionally, transcriptional post-processing, such as alternative splicing, results in a differential transcription pattern and, ultimately, physiology. Because of the composite structure of tissues, investigating transcription regulation in single cells, rather than analyzing a larger number of cells and basing result interpretation on their average behavior, is of increasing scientific interest.

The REPLI-g WTA Single Cell Kit has been specifically designed to reliably investigate effects on transcription regulation at the single-cell transcriptome level. It provides highly uniform amplification across the entire transcriptome, with negligible sequence bias. Whole transcriptome amplification from single cells that is provided by the REPLI‑g WTA Single Cell Kit complements the respective whole genome amplification kit (REPLI-g Single Cell Kit). The method is based on MDA technology, which carries out isothermal cDNA amplification utilizing a uniquely processive DNA polymerase capable of replicating up to 70 kb without dissociating from the cDNA template (see figure Multiple Displacement Amplification (MDA) technology).

Unique components of the REPLI-g WTA Single Cell Kit

All of the kit’s enzymes and amplification components undergo a unique, controlled decontamination procedure to ensure elimination of REPLI‑g amplifiable contaminating DNA or RNA. Following this process, the kits undergo stringent quality control to ensure complete functionality.

All enzymatic steps have been specifically developed to enable efficient processing of RNA for accurate amplification. For example, these processes include effective gDNA removal prior to cDNA synthesis.

Novel REPLI-g SensiPhi DNA Polymerase is used for Multiple Displacement Amplification (MDA). It is a newly developed, high-affinity enzyme that binds cDNA more efficiently, especially when the cDNA concentration is low in the reaction mixture. In addition, in contrast to PCR-based methods, REPLI-g SensiPhi DNA Polymerase has strong proofreading activity that results in 1000-fold fewer errors. It also has strong strand-displacement activity, enabling replication of cDNA through stable hairpin structures that are resistant to Taq-based whole genome or whole transcriptome amplification procedures.

Procedure

Genetic analyses often require large amounts of cDNA. Whole transcriptome amplification overcomes the limits of low RNA quantity, allowing a small number of cells, or even single cells, to be analyzed. The easy reaction setup, logical and streamlined processing steps, low handling time of just 20 minutes, and overall reaction time of just 4 hours for the complete amplification of total RNA, make the REPLI-g WTA Single Cell Kit procedure an easy and reliable method.

Lysis of cells: a single cell sample (containing 1–1000 cells) is lysed efficiently within 5 minutes, with no effect on RNA integrity. The lysed sample is used for WTA of total RNA or, optionally, mRNA-enriched (poly A+) RNA.

Generation of cDNA: following cell lysis, gDNA is removed prior to the WTA process, since accurate measurement of transcript levels depends on the elimination of false-positive results caused by gDNA contamination. Depending on the primer chosen during the subsequent reverse transcription reaction, all transcripts (if performing total RNA enrichment using random and oligo dT primers) or only poly-adenylated transcripts (if performing poly A+ mRNA enrichment using oligo dT primers) will be amplified. Consequently, the reaction will contain a mixture of random and oligo dT primers, or oligo dT primers only, which reduces rRNA amplification and ensures the 3’ ends of cDNA are reverse transcribed (transcript sizes are approximately 700–1000 bp).

Ligation: the synthesized cDNA is ligated using a high-efficiency ligation mix. Due to the nature of the subsequent ligation reaction, cDNA fragments are not assembled in the order in which they would have originally existed in the cell. However, this does not affect the detection of nucleic acid sequences, such as splicing regions, in downstream applications like NGS or qPCR.

Depending on research needs, different protocols are provided for cDNA amplification from single cells or purified RNA:

The protocol “Amplification of the 3’ Regions of mRNA (Poly A+) from Single Cells” amplifies mRNAs (and other RNAs) with poly A+ tails only and is highly suited for a wide range of applications, including NGS (RNA-Seq), real-time PCR, and microarray analysis.

The protocol “Amplification of Total RNA from Single Cells” amplifies the complete transcriptome, including RNAs with and without poly A+ tails, lnc RNAs, and linc RNAs. Note that rRNA is also amplified and will be present at a high level following amplification. If working with sequence-specific probes, such as with qPCR or arrays, the amplified rRNA will not affect downstream application results. If using the amplified RNA for RNA-Seq, be aware that more than 90% of reads are derived from rRNA; therefore sufficient reads must be obtained when performing whole-transcriptome sequencing.

The protocol “Amplification of Purified RNA” is optimized for whole transcriptome amplification from total or enriched RNA templates and is highly suited for a wide range of applications, including NGS, real-time PCR, and microarray analysis.

Applications

The REPLI-g WTA Single Cell Kit allows uniform amplification of all transcripts from very small samples, accurately representing the transcription pattern of a single cell with very limited, or no, amplification bias. Depending on the protocol, amplified cDNA is highly suited for use in next-generation sequencing (RNA-Seq), gene expression arrays, or for quantitative PCR analysis.

The REPLI-g WTA Single Cell Kit can be used for whole transcriptome amplification for analysis of: